Propargylethoxyamino nucleotide primer extensions

ABSTRACT

Propargylethoxyamino nucleosides are disclosed having the structure  
                 
 
     wherein R 1  and R 2  are —H, lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W 1  is —H or —OH; W 2  is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and W 3  is  
     —PO 4 , —P 2 O 7 , —P 3 O 10 , phosphate analog, or —OH. Additionally, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.09/583,068 filed Jun. 18, 1998, which is a division of application Ser.no. 08/696,808 filed Aug. 13, 1996, U.S. Pat. No. 5,821,356, which is acontinuation-in-part of application Ser. No. 08/694,442 filed Aug. 12,1996, abandoned.

FIELD OF THE INVENTION

[0002] This invention related generally to nucleotide compounds usefulas substrates for polymerase enzymes and polynucleotides derivedtherefrom. More specifically, this invention relates topropargylethozyamino nucleotides and their use in preparingfluorescently-labeled nucleotides useful as substrates for thermostablepolymerases, especially their use in preparing fluorescently-labelednucleotides as chain-terminating substrates in a fluorescence-based DNAsequencing method.

REFERENCES

[0003] [F]dNTP Reagents Protocol, PE Applied Biosystems, Revision A, p/n402774 March 1996)

[0004]ABI PRISM™ 373 DNA Sequencing System User's Manual, p/n 903204(June 1994)

[0005]ABI PRISM™ Dye Primer Cycle Sequencing Core Kit with AmpliTaq® DNAPolymerase, FS, Protocol, Revision C, p/n 402114 (1996)

[0006]ABI PRISM™ Dye Terminator Cycle Sequencing Core Kit Protocol, PEApplied Biosystems, Revision A, p/n 402116 (August 1995)

[0007] Benson et al., U.S. patent application Ser. No. 08/626,085 filedApr. 1, 1996

[0008] Bergot, et al, U.S. Pat. No. 5,366,860 (1994)

[0009] Connell et al., Biotechniques, 5(4):342-348 (1987)

[0010] Eckstein ed., Oligonucleotides and Analogs, Chapters 8 and 9, IRLPress (1991)

[0011] Ecketein et al., Nucleic Acids Research, 16(21):9947-59 (1988)

[0012] Gish et al., Science, 240:1520 (1988)

[0013] Hermanson, Bioconjugate Techniques, Academic Press (1996)

[0014] Hobbs, et al., U.S. Pat. No. 5,151,507 (1992)

[0015] Kasai, et al., Anal Chem., 47:34037 (1975)

[0016] Khanna, et al., U.S. Pat. No. 4,318,846 (1988)

[0017] Lee et al, Nucleic Acids Research, 20(10): 2471-2483 (1992)

[0018] Menchen et al, U.S. Pat. No. 5,188,934 (1993)

[0019] Murray, Nucleic Acids Research, 17(21): 8889 (1989)

[0020] Prober et al., Science, 238: 336-341 (1987)

[0021] Sanger, et al., Proc. Natl. Acad. Sci., 74: 5463-5467 (1977)

[0022] Scheit, Nucleotide Analogs, John Wiley (1980)

[0023] Shaw et al., Nucleic Acids Research, 23: 44954501 (1995).

[0024] Smith et al., U.S. Pat. No. 5,171,534 (1992)

[0025] Stryer, Biochemistry, W. H. Freeman (1981)

[0026] Trainor, Anal. Chem., 62: 418426 (1990)

BACKGROUND

[0027] DNA sequencing has become a vitally important technique in modernbiology and biotechnology, providing information relevant to fieldsranging from basic biological research to drug discovery to clinicalmedicine. Because of the large volume of DNA sequence data to becollected, automated techniques have been developed to increase thethroughput and decrease the cost of DNA sequencing methods (Smith;Connell; Trainor).

[0028] A preferred automated DNA sequencing method is based on theenzymatic replication technique developed by Sanger (Sanger). InSanger's technique, the DNA sequence of a single-stranded template DNAis determined using a DNA polymerase to synthesize a set ofpolynucleotide fragments wherein the fragments (i) have a sequencecomplementary to the template sequence, (ii) vary in length by a singlenucleotide, and (iii) have a 5′-end terminating in a known nucleotide,e.g., A, C, G, or T. In the method, an oligonucleotide primer isannealed to a 3′-end of a template DNA to be sequenced, the 3′-end ofthe primer serving as the initiation site for polymerase-mediatedpolymerization of a complementary polynucleotide fragment. The enzymaticpolymerization step is carried out by combining the template-primerhybrid with the four natural deoxynucleotides (“dNTPs”), a DNApolymerase enzyme, and a 2′,3′-dideoxynucleotide triphosphate (“ddNTP”)“terminator”. The incorporation of the terminator forms a fragment whichlacks a hydroxy group at the 3′-terminus and thus can not be furtherextended, i.e., the fragment is “terminated”. The competition betweenthe ddNTP and its corresponding dNTP for incorporation results in adistribution of different-sized fragments, each fragment terminatingwith the particular terminator used in the reaction. To determine thecomplete DNA sequence of the template, four parallel reactions are run,each reaction using a different ddNTP terminator. To determine the sizedistribution of the fragments, the fragments are separated byelectrophoresis such that fragments differing in size by a singlenucleotide are resolved.

[0029] In a modem variant of the classical Sanger technique, thenucleotide terminators are labeled with fluorescent dyes (Prober;Hobbs), and a thermostable DNA polymerase enzyme is used (Murray).Several advantages are realized by utilizing dye-labeled terminators:(i) problems associated with the storage, use and disposal ofradioactive isotopes are eliminated; (ii) the requirement to synthesizedye-labeled primers is eliminated; and, (iii) when using a different dyelabel for each A,G,C, or T nucleotide, all four reactions can beperformed simultaneously in a single tube. Using a thermostablepolymerase enzyme (i) permits the polymerization reaction to be run atelevated temperature thereby disrupting any secondary structure of thetemplate resulting in less sequence-dependent artifacts, and (Hi)permits the sequencing reaction to be thermocycled, thereby serving tolinearly amplify the amount of extension product produced, thus reducingthe amount of DNA template required to obtain a sequence.

[0030] While these modem variants on Sanger sequencing methods haveproven effective, several problems remain with respect to optimizingtheir performance and economy. One problem encountered when usingdye-labeled terminators in combination with thermostable polymeraseenzymes, particularly in the case of fluorescein-type dye labels, isthat a large excess of dye-labeled terminator over the unlabeled dNTPsis required, up to a ratio of 50:1. This large excess of labeledterminator makes it necessary to purify the sequencing reaction productsprior to performing the electrophoretic separation step. This clean-upstep is required in order to avoid interference caused by thecomigration of unincorporated labeled terminator species and bona fidesequencing fragments. A typical clean-up method includes an ethanolprecipitation or a chromatographic separation (ABI PRISM™ Dye TerminatorCycle Sequencing Core Kit Protocol). Such a clean-up step greatlycomplicates the task of developing totally automated sequencing systemswherein the sequencing reaction products are transferred directly intoan electrophoretic separation process. A second problem encountered whenusing presently available dye-labeled terminators in combination with athermostable polymerase is that an uneven distribution of peak heightsis obtained in Sanger-type DNA sequencing.

SUMMARY

[0031] The present invention is directed towards our discovery of anovel class of propargylethoxyamino nucleotides useful aschain-terminating dideoxynucleotides, and, as chain-extendingdeoxynucleotides, in a primer extension reaction, e.g., in a Sanger-typeDNA sequencing or in a PCR reaction.

[0032] It is an object of the invention to provide a nucleotide whichcan be used to form a labeled chain-terminating nucleotide.

[0033] It is a further object of the invention to provide achain-terminating nucleotide which includes a label.

[0034] It is yet an additional object of the invention to provide achain-terminating nucleotide which includes a fluorescent label whereina reduced excess concentration of such labeled chain-terminatingnucleotide over an unlabeled chain-terminating nucleotide is required ina Sanger-type DNA sequencing process.

[0035] It is another object of the invention to provide a labeledchain-terminating nucleotide which results in a more even distributionof peak heights in a Sanger-type DNA sequencing process.

[0036] It is an object of the invention to provide a nucleotide whichcan be used to form a labeled chain-extending deoxynucleotide.

[0037] It is a further object of the invention to provide achain-extending deoxynucleotide which includes a label.

[0038] It is an additional object of the invention to provide methodsincluding a primer extension reaction utilizing the propargylethozyaminonucleotides of the invention.

[0039] In a first aspect, the foregoing and other objects of theinvention are achieved by a nucleoside compound having the structure:

[0040] wherein the variable substituents R₁-R₂ and W₁-W₃ are defined asfollows. R₁ and R₂ taken separately are —H, lower alkyl protectinggroup, or label. In a preferred embodiment, one of R₁ and R₂ is label,the label preferably being a fluorescein-type dye or a rhodamine-typedye. B is a 7-deazapurine, purine, or pyrimidine nucleoside base,preferably uracil cytosine, 7-deazadenine, or 7-deazaguansine. When B ispurine or 7-deazapurine, the sugar moiety is attached at the N⁹-positionof the purine or deazapurine, and when B is pyrimidine, the sugar moietyis attached at the N¹-position of the pyrimidine. When B is a purine,the adjacent triple-bonded carbon is attached to the 8-position of thepurine, when B is 7-deazapurine, the adjacent triple-bonded carbon isattached to the 7-position of the 7-deazapurine, and when B ispyrimidine, the adjacent triple-bonded carbon is attached to the5-position of the pyrimidine. W₁ is —H or —OH. W₂ is —OH or a moietywhich renders the nucleoside incapable of forming a phosphodiester bondat the 3′-position. W₃ is —PO₄, —P₂O₇, —P₃O₁₀, phosphate analog, or —OH.

[0041] In a second aspect, the invention includes a method forperforming a primer extension reaction including the following steps:providing a template nucleic acid; annealing an oligonucleotide primerto a portion of the template nucleic acid; and adding primer-extensionreagents to the primer-template hybrid for extending the primer. In animportant aspect of the invention, the primer extension reagents includea propargylethoxyamino nucleoside compound having the structuredescribed above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIGS. 1A-1C show results from a Terminator Titration Assay usinga variable concentration of dye-labeled terminator.

[0043]FIG. 2 shows a comparison of sequencing patterns obtained usingconventional dye-labeled terminators and dye-labeled terminators of theinvention.

[0044]FIG. 3 shows a diagram of the Single Nucleotide IncorporationAssay used to characterize the dye-labeled terminators of the invention.

[0045]FIG. 4 shows results from the Single Nucleotide IncorporationAssay comparing the rates of incorporation of unlabeled and dye-labeledterminators.

[0046]FIG. 5 shows the synthesis of 2-phthalimidoethanol (3) and3-(2-phthalimidoethoxy)propyne (5).

[0047]FIG. 6 shows the synthesis of5-{3-(2-phthalamidoethoxy)-propyn-1-yl}-2′,3′-dideoxycytidine (7) and of5-{3-(2-trifluoroacetamidoethoxy)propyn-1-yl}-2′,3′-dideoxycytidine (8).

[0048]FIG. 7 shows the synthesis of5-{3-(2′-trifluoroacetadoethoxy)propyn-1-yl}-2′,3′-dideoxycytidinemonophosphate (10).

[0049]FIG. 8 shows the synthesis of5-{3-(2-trifluoroacetamidoethoxy)propyn-1-yl}-2′,3′-dideoxycytidinetriphosphate (12) and5-{3-(2-aminoethoxy)propyn-1-yl}-2′,3′-dideoxycytidine triphosphate(13).

[0050]FIG. 9 shows the synthesis of 2-(2-phthalimidoethoxy)ethanol (15)and 3-[2(2-phthalimidoethoxy)ethoxy]propyne (16).

[0051]FIG. 10 shows the synthesis of5-[3-{2-(2-phthalamidoethoxy)ethoxy}propyn-1-yl]-2′,3′-dideoxycytidine(17) and5-[3-{2-(2-trifuoroacetamidoethoxy)ethoxy)propyn}-1-yl]-2′,3′-dideoxycytidine(18).

[0052]FIG. 11 shows the synthesis of5-[3-{2-(2-trifluoroacetamidoethoxy)-ethoxy}propyn-1-yl]-2′,3′-dideoxycytidinemonophosphate (20).

[0053]FIG. 12 shows the synthesis of5-[3-{2-(2-trifuoroacetamidoethoxy)ethoxy}propyn-1-yl]-2′,3′)-dideoxycytidinetriphosphate (22) and5-[3-{2(2-aminoethoxy)ethoxy}propyn-1-yl]-2′,3′-dideoxycytidinetriphosphate (23).

[0054]FIG. 13 shows results from a single color sequencing reactionusing DTAMRA-1-labeled terminator including a propargylamido linker(top), and DTAMRA-2-labeled terminator including apropargyl-1-ethoxyamido linker (bottom).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Reference will now be made in detail to the preferred embodimentsof the invention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that they are not intendedto limit the invention to those embodiments. On the contrary, theinvention is intended to cover alternatives, modifications, andequivalents, which may be included within the invention as defined bythe appended claims.

[0056] Generally, the present invention comprises a novel class ofpropargylethoxyamino nucleoside compounds useful as substrates forpolymerase enzymes. The compounds of the present invention findparticular application as labeled dideoxynucleotide chain-terminatingagents for use in Sanger-type DNA sequencing methods, and, as labeleddeoxynucleotide chain-ending agents for use in methods including aprimer extension reaction, e.g., PCR.

[0057] The invention is based in part on the discovery that the subjectdye-labeled nucleotides are particularly good substrates forthermostable DNA polymerase enzymes, e.g., a significantly reduced molarexcess is required in a Sanger-type DNA sequencing reaction relative tothat required when using currently available dye-labeled terminators.

I. DEFINITIONS

[0058] Unless stated otherwise, the following terms and phrases as usedherein are intended to have the following meanings:

[0059] The term “lower alkyl” denotes straight-chain and branchedhydrocarbon moieties containing from 1 to 8 carbon atoms, i.e., methyl,ethyl, propyl, isopropyl, tert-butyl, isobutyl sec-butyl neopentyl,tert-pentyl, and the like.

[0060] The term “label” refers to a moiety that, when attached to thenucleosides of the invention, render such nucleosides, andpolynucleotides containing such nucleotides, detectable using knowndetection means. Exemplary labels include fluorophores, chromophores,radioisotopes, spin-labels, enzyme labels, chemiluminescent labels, andthe like, which allow direct detection of a labeled compound by asuitable detector, or, a ligand, such as an antigen, or biotin, whichcan bind specifically with high affinity to a detectable anti-ligand,such as a labeled antibody or avidin. Preferably the labels arefluorescent dyes such as fluorescein-type or rhodamine-type dyes (Lee;Menchen).

[0061] The term “nucleoside” refers to a compound consisting of apurine, deazapurine, or pyrimidine nucleoside base, e.g., adenine,guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, andthe like, linked to a pentose at the 1′ position, including 2′-deoxy and2′-hydroxyl forms (Stryer). The term “nucleotide” as used herein refersto a phosphate ester of a nucleoside, e.g., triphosphate esters, whereinthe most common site of esterification is the hydroxyl group attached atthe C-5 position of the pentose. Many times in the present disclosurethe term nucleoside will be intended to include both nucleosides andnucleotides. “Analogs” in reference to nucleosides include syntheticanalogs having modified base moieties, modified sugar moieties, and/ormodified phosphate ester moieties, e.g., as described elsewhere (Scheit;Eckstein 1991).

[0062] As used herein, the terms “polynucleotide” or “oligonucleotide”refer to linear polymers of natural nucleotide monomers or analogsthereof including double and single stranded deoxyribonucleotides,ribonucleotides, α-anomeric forms thereof and the like. Usually thenucleoside monomers are linked by phosphodiester linkages, where as usedherein, the term “phosphodiester linkage” refers to phosphodiester bondsor bonds including phosphate analogs thereof, including associatedcounterions, e.g., H, NH₄, Na, and the life if such counterions arepresent. Polynucleotides typically range in size from a few monomericunits, e.g. 8-40, to several thousands of monomeric units. Whenever apolynucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′->3′order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytidine, “G” denotes deoxguanosine, and “T” denotesthymidine, unless otherwise noted.

[0063] The term “phosphate analog” refers to analogs of phosphatewherein the phosphorous atom is in the +5 oxidation state and one ormore of the oxygen atoms is replaced with a non-oxygen moiety, exemplaryanalogs including phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, boronophosphates, and the like,including associated counterions, e.g., H, NH₄, Na, and the like if suchcounterions are present.

[0064] As used herein, the term “propargylamido linker” shall refer to alinker having the structure

—C≡C—CH₂—NH—

[0065] the term “propargyl-1-ethoxyamido linker” shall refer to a linkerhaving the structure

—C≡C—CH₂—O—CH₂CH₂—NH—

[0066] and the term “propargyl-2-ethoxyamido linker ” shall refer to alinker having the structure

—C≡C—CH₂-(O—CH₂-CH₂)₂—NH—

[0067] where, for each of the above structures, the terminal end of theacetylene is bound to a nucleotide base, and the amide nitrogen is boundthrough a convenient linkage to a label.

[0068] The term “fluorescein-type dyes” refers to a class of xanthenedye molecules which include the following fused three-ring system:

[0069] where a wide variety of substitutions are possible at each deoxyring position. A particularly preferred subset of fluorescein-type dyesinclude the 4,7,-dichorofluoresceins (Menchen). Examples offluorescein-type dyes used as fluorescent labels in DNA sequencingmethods include 6-carboxyfluorescein (6-FAM), 5-carboxyfluorescein(5-FAM), 6-carboxy-4,7,2′,7′-tetrachlorofluorescein (TET),6-carboxy4,7,2′,4′,5′,7′-hexachlorofluorescein (HEX), 5-(and6)carboxy4′,5′-dichloro-2′7′-dimethoxyfluorescein (JOE), and5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein (ZOE). Many times thedesignation -1 or -2 is placed after an abbreviation of a particulardye, e.g., HEX-1. The “−1” and “−2” designations indicate the particulardye isomer being used. The 1 and 2 isomers are defined by the elutionorder (the 1 isomer being the first to elute) of free dye in areverse-phase chromatographic separation system utilizing a C-8 columnand an elution gradient of 15% acetonitrile/85% 0.1 M triethylammoniumacetate to 35% acetonitrile/65% 0.1 M triethylammonium acetate,.

[0070] The term “rhodamine-type dyes” refers to a class of xanthene dyemolecules which include the following fused three-ring system:

[0071] where preferably Y₁-Y₄ taken separately are hydrogen or loweralkyl, or, when taken together, Y₁ and R₂ is propano and Y₂ and R₁ ispropano, or, when taken together, Y₃ and R₃ is propano and Y₄ and R₄ ispropano. A wide variety of substitutions are possible at each deoxy ringposition including the R₁-R₄ positions. Exemplary rhodamine type dyesuseful as nucleoside labels include tetramethylrhodamine (TAMRA),4,7-diclorotetramethyl rhodamine (TAMRA), rhodamine X (ROX), rhodamine6G (R₆G), rhodamine 110 (R110), and the like (Bergot; Lee).

[0072] As used herein, the term “FLAN dyes” referes to asmmetricbenzoxanthene dye compounds having the formula;

[0073] wherein Y₁ and Y₂ taken separately are hydroxyl, oxygen,imminium, or amine. R₁-R₄ taken separately are hydrogen, fluorine,chlorine, lower alkyl, lower alkene, lower alkyne, safonate, amino,ammonium, amido, nitrile, alkoxy, linking group, or combinations thereofAnd, R₉ is acetylene, alkane, alkene, cyano, substituted phenyl, orcombinations thereof the substituted phenyl having the structure:

[0074] wherein X₁ is carboxylic acid or sulfonic acid; X₂ and X₅ takenseparately are hydrogen, chlorine, fluorine, or lower alky; and X₃ andX₄ taken separately are hydrogen, chlorine, fluorine, lower alkylcarboxylic acid, sulfonic acid, or linking group (Benson).

[0075] As used herein the term “primer-extension reagent” means areagent including components necessary to effect the enzymatictemplate-mediated extension of an oligonucleotide primer. Primerextension reagents include: (i) a polymerase enzyme, e.g., athermostable polymerase enzyme such as Taq polymerase; (ii) a buffer;(iii) deoxynucleotide triphosphates, e.g., deoxyguanosine5′-triphosphate, 7-deazadeoxyguanosine 5′-triphosphate, deoxyadenosine5′-triphosphate, deoxythymidine 5′-triphosphate, deoxycytidine5′-triphosphate; and, optionally in the case of DNA sequencingreactions, (iv) dideoxynucleotide triphosphates, e.g., dideoxyguanosine5′-triphosphate, 7-deazadideoxyguanosine 5′-triphosphate,dideoxyadenosine 5′-triphosphate, dideoxythymidine 5′-triphosphate, anddideoxycytidine 5′-triphosphate.

[0076] “Template nucleic acid” refers to any nucleic acid which can bepresented in a single stranded form and is capable of annealing with aprimer oligonucleotide. Exemplary template nucleic acids include DNA,RA, which DNA or RNA may be single stranded or double stranded. Moreparticularly, template nucleic acid may be genomic DNA, messenger RNA,cDNA, DNA amplification products from a PCR reaction, and the like.Methods for preparation of template DNA may be found elsewhere (ABIPRISM™ Dye Primer Cycle Sequencing Core Kit).

II. PROPARGYLETHOXYAMINO NUCLEOTIDE COMPOUNDS

[0077] In a first aspect, the present invention comprises a novel classof propargylethoxyamino nucleoside compounds having the generalstructure shown immediately below as Formula I. (Note that all molecularstructures provided throughout this disclosure are intended to encompassnot only the exact electronic structure presented, but also include allresonant structures and protonation states thereof.)

[0078] Referring to Formula I, R₁ and R₂ are chosen from among, —H,lower all, protecting group, or label. Preferably, the label is afluorescent dye. More preferably the label is a fluorescein-typefluorescent dye or a rhodamine-type fluorescent dye. Preferably, whenone of R₁ and R₂ is a label, the other is either —H or lower alkyl.Preferred protecting groups include acyl, alkoxycarbonyl or sulfonyl.More preferably, the protecting group is trifluoroacetyl.

[0079] The label is attached to the nucleoside through a“linkage”-typically formed by the reaction of the primary or secondaryamino moiety of the propargylethoxyamino nucleoside with a“complementary functionality” located on the label. Preferably, thecomplementary functionality is isothiocyanate, isocyanate, acyl azide,N-hydroxysuccinimide (NHS) ester, sulfonyl chloride, aldehyde orglyoxal, epoxide, carbonate, aryl halide, imidoester, carbodiimide,anhydride, 4,6-dichlorotriazinylamine, or other active carboxylate(Hermanson). In a particularly preferred embodiment, the complementaryfunctionality is an activated NHS ester which reacts with the amine ofthe propargylethoxyamino nucleoside of the invention, where to form theactivated NHS ester, a label including a carboxylate complementaryfunctionality is reacted with dicyclohexylcarbodiimide andN-hydroxysuccinimide to form the NHS ester (Khanna, Kasai). Table 1below shows a sampling of representative complementary functionalitiesand resulting linkages formed by reaction of the complementaryfunctionality with the amine of the propargylethoxyamino nucleoside.TABLE 1 Complementary Functionality Linkage —NCS —NHCSNH—

—SO₂X —SO₂NH—

[0080] Again referring to Formula I, B is a 7-deazapurine, purine, orpyrimidine nucleotide base, where in a preferred embodiment, B is chosenfrom the group consisting of uracil, cytosine, 7-deazaadenine, and7-deazaguanosine. When B is purine or 7-deazapurine, the sugar moiety ofthe nucleotide is attached at the N⁹-position of the purine ordeazapurine, and when B is pyrimidine, the sugar moiety is attached atthe N¹-position of the pyrimidine. When B is a purine, the adjacenttriple-bonded carbon is attached to the 8-position of the purine, andwhen B is 7-deazapurine, the adjacent triple-bonded carbon is attachedto the 7-position of the 7-deazapurine, and when B is pyrimidine, theadjacent triple-bonded carbon is attached to the 5-position of thepyrimidine.

[0081] W₁ is selected from among —H and —OH. When W₁ is —OH thenucleoside is a ribonucleotide, and when W₁ is —H the nucleoside is adeoxyribonucleotide.

[0082] W₂ is —OH or a moiety which renders the nucleoside incapable offorming a phosphodiester bond at the 3′-position. Preferred moietiesuseful for this function include —H, azido, amino, fluro, methoxy, andthe like.

[0083] W₃ is selected from the group consisting of —PO₄, —P₂O₇, —P₃O₁₀,phosphate analog, and —OH. In a preferred embodiment useful forenzymatic synthesis of polynucleotides, W₃ is —P₃O₁₀.

[0084] Generally, the propargylethoxyamino nucleosides of the inventionare prepared as follows. Bromoethanol is reacted with potassiumphthalimide to give a phthalimido derivative. The phthalimido derivativeis then O-alkylated with propargyl bromide in the presence of NaH,resulting in a protected 3-(2-phthalamidoethoxy)propyne linking arm. Aniodo-nucleoside is then reacted with the protected linking arm in thepresence of cuprous iodide, tetrakis(triphenylphosphine)palladium, andtriethylamine in dimethylformamide for approximately 12 hours at ambienttemperature or until the reaction is complete as determined by TLC. Thesolution is then concentrated in vacuo and the product is purified bysilica gel flash chromatography and is analyzed for identity and purifyby proton NMR and analytical reverse-phase HPLC (C-18 column). Treatmentwith ethylenediamine, followed by acetylation with ethyltrifluoroacetate, gave a nucleoside-linking arm compound. Freshlydistilled phosphorous oxychloride is added to the nucleoside-linking armcompound in trimethylphosphate at −30° C. to form the correspondingdichloromonophosphate. The reaction mixture is quenched with 2 Mtetraethylammonium bicarbonate (TEAB) pH 8.0 to yield the monophosphate,which is then purified by preparative reverse-phase (C-18 column). Themonophosphate is activated with carbonyldiimidazole (CDI) and excess CDIis quenched with MeOH. The activated monophosphate is reacted, at roomtemperature, with tributylammonium pyrophosphate. When complete, thereaction is quenched with 0.2 M TEAB and purified by reverse phase BPLC(C-18 column). The purified protected triphosphate is evaporated todryness and resuspended in concentrated aqueous NH₁₄OH to remove the TFAgroup. The deprotected triphosphate solution is evaporated to drynessand formulated with 0.1 M TEAB pH 7.0 to a desired concentration. Theconcentration and purity of the formulated bulk are confirmed by UT/VVisspectroscopy and ion-pairing HPLC respectively.

[0085] Generally, in a preferred method, dye-labeledpropargylethoxyamino nucleosides of the invention are prepared asfollows. A propargylethoxyamino nucleoside is dissolved in 100 mM TEAB(pH 7.0), the solution is evaporated to dryness, and the nucleoside isresuspended in 250 mM sodium bicarbonate buffer (pH 9.0). Dye-NHS (inDMSO) is added and allowed to react overnight with stirring. Whencomplete, the reaction mixture is purified by an ion exchange andreverse phase HPLC (C-18 column). The dye labeled triphosphatenucleotide solution is evaporated to dryness and formulated with 50 mM3-[cyclohexylaminol-2-hydroxy-1]-propane-sulfonic acid (CAPSO) pH 9.6 toa desired concentration. The concentration and purity of the formulatedbulk are confirmed by UV/Vis spectroscopy and ion-pairing HPLC,respectively.

III. METHODS UTILIZING THE PROPARGYLETHOXYAMINO COMPOUNDS

[0086] The propargylethoxyamino compounds of the invention areparticularly well suited for use in methods which include atemplate-mediated primer extension reaction of the type including thefollowing steps: (i) providing a template nucleic acid; (ii) annealingan oligonucleotide primer to a portion of the template nucleic acidthereby forming a primer-template hybrid; and (iii) addingprimer-extension reagents to the primer-template hybrid for extendingthe primer. In particular, the compounds of the invention provide ameans for incorporating a label directly into a primer extensionproduct.

[0087] In a first preferred class of methods utilizing a primerextension reaction, the extension products are labeled by includinglabeled deoxynucleotide triphosphates or deoxyribonucleosidetriphosphates of the invention into the primer extension reactionthereby randomly incorporating labels throughout the extension product([F]dNTP Reagents Protocol). Such a method can be used to label PCRamplicons as well as single-primer derived extension products. To labelan extension product in this way, the primer extension reaction isperformed using established protocols, but a labeled deoxynucleotidetriphosphate is added to the reaction. Generally, to perform a primerextension reaction in the context of PCR, template nucleic acid is mixedwith 20 pmol of each primer and primer-extension reagents comprising 20mM buffer at pH 8, 1.5 mM MgCI₂, 50 mM of each deoxynucleotidetriphosphate (dNTP), and 2 units of Taq polymerase or other suitablethermostable polymerase. The reaction mixture is then thermocycled, atypical thermocycle profile comprising a denaturation step (e.g. 96° C.,15 s), a primer annealing step (e.g., 55° C., 30 s), and a primerextension step (e.g., 72° C., 90 s). Typically, the thermocycle isrepeated from about 10 to 40 cycles. For PCR amplifications, the typicalratio of labeled deoxynucleotide triphosphate to unlabeleddeoxynucleotide triphosphate is between 100:1 to 1000:1, depending onthe amount of signal desired. The maximum ratio of labeleddeoxynucleotide triphosphate to unlabeled deoxynucleotide triphosphatethat can be used in a PCR reaction mixture without adversely affectingamplification efficiency is approximately 1:4.

[0088] In a second preferred class of methods utilizing a primerextension reaction, the extension products are labeled by includinglabeled dideoxynucleotide triphosphates or dideoxyribonucleosidetriphosphates of the invention into the primer extension reactionthereby randomly incorporating detectable labels at the 3′-terminalnucleotide, e.g., Sanger-type DNA sequencing. Generally, to perform aprimer extension reaction in the context of Sanger-type DNA sequencingusing labeled dideoxynucleotide triphosphates of the invention, 1 μl oftemplate solution (1 ml of PCR reaction diluted with 5 ml water) and 2μl of primer (0.4 pmol/μl) is mixed with primer-extension reagentscomprising 2 μl buffer (400 mM Tris-HCl, 10 mM MgCl₂, pH 9.0.), 2 μl ofa deoxynucleotide/labeled dideoxynucleotide mixture T-terminationreaction, 1250 μM ddTTP, 250 μM dATP, 250 μM dCTP, 180 μM7-deaza-dGTP,and 250 μM dTTP), and 2 μl of polymerase enzyme (5 Units/μl where oneunit is defined as in Lawyer). The reaction is then thermocycled usingthe following exemplary program: denaturation at 98° C. for 5 s followedby repeated cycles of 96 ° C. for 5 s; 55 ° C. for 40 s; 68° C. for 1min, where the cycle is repeated approximately 15 times.

[0089] The propargylethoxyamino compounds of the invention may also beused in the context of variants of Sanger-type sequencing methods whichrely on base-specific cleavage of the primer extension products, e.g.,methods utilizing labile nucleotides (Eckstein 1988; Shaw).

IV. EXAMPLES

[0090] The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of theinvention and not to in any way limit its scope.

Example 1 Terminator Titration Assay for Determining the RequiredTerminator Excess in a Sequencing Reaction

[0091] The Terminator Titration Assay was used to determine the minimumamount of dye terminator required to create a full sequencing ladder,i.e., a sequencing ladder including all fragments terminating in aparticular base having a length of between about 20 to about 600nucleotides. The key components of the assay were (i) a primer labeledwith a first dye, and (ii) a terminator labeled with a second dyespectrally resolvable from the first dye. In the assay, when aninsufficient concentration of dye terminator was added to the sequencingreaction, no dideoxy-terminated fragments were formed, and all that wasseen on the sequencing gel were products formed by “false stops” thatwere labeled with the first dye only. As used herein the term “falsestops” refer to primer extension products not terminating in a dideoxyterminator, such products probably being formed when the polymeraseenzyme spontaneously disengages with the template strand. When too muchterminator was used, only short termination products were formed, i.e.,less than about 50 nucleotides in length, such products including boththe first and second dyes. When the proper amount of terminator wasused, a full sequencing ladder was produced, each fragment of the ladderbeing labeled with both the first and second dyes.

[0092] The dye-terminator reactions were performed using AmpliTaq DNAPolymerase, FS following protocols provided in the ABI PRISM™ DyeTerminator Cycle Sequencing Core Kit Manual (PE Applied Biosystems pin402116). (The FS enzyme is a recombinant Thermus aquaticus DNApolymerase having two point mutations--G46D and F667Y). All reagentsexcept the dNTP mix, dye labeled primers, and dye-labeled terminatorswere from an ABI PRISM™ Dye Terminator Core Kit (PE Applied Biosystemsp/n 402117). The dNTP mix consisted of 2 mM each of DATP, dCTP, dGTP anddTTP. A premix of reaction components was prepared as shown in thefollowing table wherein all quantities are given on a per reactionbasis: 5X Buffer 4.0 μL dNTP mix 1.0 μL Template: pGEM ®-3Zf(+), 0.2μg/μL 5.0 μL Primer: −21 M13 (forward), 0.3 pmol/μL 4.0 μL AmpliTaq DNAPolymerase, FS 0.5 μL H₂O 0.5 μL

[0093] Reactions were assembled in 0.5 ml tubes adapted for thePerkin-Elmer 480 DNA Thermal Cycler (PE Applied Biosystems p/nNSO1-100). Reaction volumes were 20 μl, including 15 μL of the abovereaction premix, a variable amount of dye labeled terminator, and asufficient volume of water to bring the total react-ion volume up to 20μl. From 1 to 1000 pmol of the dye terminator was added to eachreaction. 30 μl of mineral oil was added to the top of each reaction toprevent evaporation. Reactions were thermocycled as follows: 96° C. for30 sec, 50° C. for 15 sec, and 60° C. for 4 min, for 25 cycles; followedby a 4° C. hold cycle.

[0094] All reactions were purified by spin-column purification onCentri-Sep spin columns according to manufacturer's instructions(Princeton Separations p/n CS-901). Gel material in the column washydrated with 0.8 mL deionized water for at least 30 minutes at roomtemperature. After the column was hydrated and it was determined that nobubbles were trapped in the gel material, the upper and lower end capsof the column were removed, and the column was allowed to drain bygravity. The column was then inserted into the wash tubes provided inthe kit and centrifuged in a variable speed microcentrifuge at 1300×gfor 2 minutes, removed from the wash tube, and inserted into a samplecollection tube. The reaction mixture was carefully removed from underthe oil and loaded onto the gel material. Columns were centrifuged in avariable speed microcentrifuge at 1300×g for 2 minutes. Eluted sampleswere then dried in a vacuum centrifuge.

[0095] Prior to loading onto a sequencing gel, the dried samples wereresuspended in 25 μL of Template Suppression Reagent (PE AppliedBiosystems p/n 401674), vortexed, heated to 95° C. for 2 minutes, cooledon ice, vortexed again, and centrifuged (1300×g). 10 μL of theresuspended sample was aliquoted into sample vials (PE AppliedBiosystems p/n 401957) adapted for the PE ABI PRISM™ 310 GeneticAnalyzer (PE Applied Biosystems p/n 310-00-100/120). Electrophoresis onthe 310 Genetic Analyzer was performed with sieving polymers andcapillaries specially adapted for DNA sequencing analysis (PE AppliedBiosystems pin 402837 (polymer) and p/n 402840 (capillary), or, p/n402091 (polymer) and p/n 401821 (capillary)). In each case, the sievingpolymer included nucleic acid denaturants. Samples wereelectrokinetically injected onto the capillary for 30 sec at 2.5 kV, andrun for 2 hr at 10 to 12.2 kV with the outside wall of the capillarymaintained at 42° C.

[0096] FIGS. 1A-C show typical results from a Terminator Titration Assaycollected on the 310 analyzer. In each case, the dye-labeled terminatoremploys the traditional propargylamido linker. The traces showfluorescence intensity at a given wavelength as a function of timeduring an electrophoresis run for nucleotides 71-175. The amount ofdye-terminator added to the primer extension reaction was variable,where in FIG. 1A 1 pmol terminator was used, in FIG. 1B 4 pmolterminator was used, and in FIG. 1C 150 pmol terminator was used. Thetop trace in each panel is fluorescence emitted by the dye-labeledterminator and collected at 535-545 nm and the bottom trace in eachpanel is fluorescence emitted from the dye-labeled primer and collectedat 575-585 mm. The dye primer trace (bottom) shows false stops, i.e.,fragments not terminating in a dye-labeled terminator, as well asproperly terminated fragments. False stops occur when there isinsufficient terminator or when a terminator is a poor polymerasesubstrate. The dye terminator trace (top) shows the specificincorporation of the dye-labeled terminator. The experimental conditionswere as follows: Terminator: ddATP labeled with 6-FAM at variableconcentration Primer: TAMRA labeled −21M13 (forward) Template:pGEM-3Zf(+) DNA Polymerase: AmpliTaq ® DNA Polymerase, FS.

[0097]FIG. 1A shows data for a reaction using 1 pmol 6-FAM-ddATP. Verylittle specific incorporation was detected as evidenced by the smallpeaks in the dye terminator trace. The false stops shown in the bottomdye-primer trace were essentially as large as anyspecifically-terminated peaks. This pattern indicates that thedye-terminator concentration was too low. FIG. 1B shows data for areaction using 4 pmol 6-FAM-ddATP. Good specific terminatorincorporation was observed with relatively even peak heights throughoutthe sequencing ladder. In the dye primer trace, easily distinguishablepeaks above the false-stop noise were present, the peaks comigratingwith the peaks in the dye terminator trace. This pattern indicates thatthe dye terminator concentration was within a useable range. FIG. 1Cshows data for a reaction using 150 pmol 6-FAM-ddATP. A “top heavy”pattern was seen with the early peaks showing very high levels of dyeterminator incorporation and the later peaks showing much lower levelsof incorporation. This pattern indicates that the dye-terminatorconcentration was too high.

[0098]FIG. 2 shows a comparison of the sequencing patterns obtained for-6-FAM-ddCTP terminators using the propargylamido linker at aconcentration of 250 pmol (top trace) and for 6-FAM-ddCTP terminatorsusing the propargyl-1-ethoxyamido linker of the invention at aconcentration of 50 pmol (bottom trace). These concentrations weredetermined to be the optimal concentration for each type of terminator.For the 6-FAM-ddCTP terminator using the propargylamido linker, the meanpeak height was 1800, and the standard deviation was 959, resulting in arelative error of 0.533. For the 6-FAM-ddCTP terminator using thepropargyl-1-thoxyamido linker of the invention, the mean peak height was504, and the standard deviation was 220, resulting in a relative errorof 0.436. The lower relative error obtained when using thepropargyl-1-ethoxyamido linker indicates a more even peak heightdistribution, which facilitates base-calling in an automated DNAsequencing system.

Example 2 Amount of FAM-Labeled C-Terminator Required to Form a FullSequencing Ladder as a Function of Linker Type

[0099] The table below shows the relative molar excess of dye-labeledC-terminator required to form a full sequencing ladder according to theTerminator Titration Assay as described above in Example 1. The relativemolar excess is defined such that the amount of unlabeled dideoxyterminator required to form a full sequencing ladder results in a valueof 1. In each case a C-terminator was linked to a 6-FAM dye. As can beseen from the table, the C-terminator employing thepropargyl-1-ethoxyamido linker requires a six-fold reduced molar excessas compared with terminators employing the traditional propargylamidolinker (9 vs 55) and a five-fold reduced molar excess as compared withterminators employing a propargyl-2etoxyamido linker (9 vs 45).^(a)Relative Molar Excess Terminator Linker Arm Required Unlabeledterminator  1 Propargylamido 55 Propargyl-1-  9 ethoxyamido Propargyl-2-45 ethoxyamido

[0100] a The relative molar excess is defined such that the amount ofunlabeled dideoxy terminator required to form a full sequencing ladderresults in a value of 1.

Example 3 Relative Molar Excess of FAM-Labeled C-Terminator Required toForm a Full Sequencing Ladder as a Function of Linker Type and Dye

[0101] The table below compares the relative molar excess of dye-labeledC-terminator required to form a fill sequencing ladder according to theTerminator Titration Assay as described above in Example 1 for variouscombinations of dyes and linkers. The relative molar excess is definedas above. As can be seen from the table, the C-terminator employing thepropargyl-1-thoxyamido linker results in from a six-fold to a two-foldreduction in molar excess as compared with terminators includingexisting propargylamido linkers, depending on the particular dye used.Relative Molar Excess Linker Type Dye Terminator Required None None 1Propargylamido 6-FAM 55 Propargyl-1- 6-FAM 9 ethoxyamido PropargylamidoHEX-2 >250 Propargyl-1- HEX-2 45 ethoxyamido Propargylamido HEX-1 25Propargyl-1- HEX-1 12 ethoxyamido Propargylamido FLAN-2 60 Propargyl-1-FLAN-2 12 ethoxyamido Propargylamido TET-2 60 Propargyl-1- TET-2 20ethoxyamido

Example 4 Single Nucleotide Incorporation Assay for Measuring RelativeEnzyme Selectivity

[0102] A. General Description of the Assay

[0103] The assay described in this Example measures the preference thata DNA polymerase shows for a non-dye-labeled terminator over adye-labeled terminator for the purpose of quantifying the effect ofdifferent terminator-dye linker arm structures on terminatorincorporation. In the assay, an unlabeled terminator and its cognatedye-labeled terminator are present in equal concentrations and allowedto compete for the same polymerase-substrate binding site under enzymelimited (steady-state) conditions.

[0104] With reference to FIG. 3, the components of the assay include a36 nucleotide template (5); a 25 nucleotide primer (10) having asequence complementary to the template and a first fluorescent label atthe 5′-end (15); an unlabeled terminator (20); a dye-labeled terminator(25) having a second fluorescent label attached thereto (30) which isspectrally resolvable from the first fluorescent label; and a polymeraseenzyme. In the present example, the unlabeled terminator was 2′,3′-ddCTPand the dye-labeled terminator was 6-(FAM-ddCTP where different linkerarms were used to attach the dye to the nucleotide. The template DNAcontained a single G (35) at the template position following the end ofthe primer. Incorporation of the unlabeled terminator (20) resulted inthe formation of a 26-base long primer-product ending in ddC (40), whileincorporation of the dye-labeled terminator resulted in the formation ofa 26-base long primer-product ending with a (FAM)-ddC (45).

[0105] Products (40) and (45) were detected by resolving themelectrophoretically and detecting the resulting bands using an ABIPRISM™ 373 DNA Sequencer (PE Applied Biosystems). A typical bandingpattern consisted of a 25-mer band corresponding to the labeled primer(10), a 26-mer band corresponding to the product (40) and an “apparent27-mer” which corresponded to product (45). The apparent extra base seenin product (45) is a result of the effect of the dye-labeled terminatoron the electrophoretic mobility of the fragment. By taking samples froma reaction mixture and measuring the relative amounts of DNA in the25-mer, 26-mer, and apparent 27-mer bands as a function of time, it waspossible to measure the relative incorporation rates of the unlabeledand dye-labeled terminators in the reaction. After correcting for dyeenergy transfer (see below), the ratio of rates of incorporation wasfound to be a direct measure of the enzyme's preference for an unlabeledterminator over a dye-labeled terminator. In this manner, it waspossible to measure the effect of linker structure on dye-terminatorincorporation.

[0106] B. Energy Transfer Correction

[0107] For the case of product (45), it is necessary to correct thefluorescence signal for fluorescence energy transfer which takes placebetween the first fluorescent label (15) located at the 5′-end ofproduct (45), TAMRA in this example, and the second fluorescent label(30) located on the terminator positioned at the 3′-end of product (45),FAM in this example. Under the conditions used for detection, thepresence of the 3′-FAM label serves to enhance the signal resulting fromthe TAMRA label. The energy-transfer correction is accomplished bysynthesizing a doubly labeled internal standard molecule having the samestructure as product (45), preparing a control sample containing equalmoles of the 5′-TAMRA labeled primer (10) and the doubly labeledinternal standard molecule, and running the control sample in a controllane on the same gel as assay products (40) and (45). Any difference inTAMRA fluorescence between the primer (10) and the doubly labeledinternal standard is a quantitative measure of the extent of energytransfer between the dye moieties in the assay product (45). Forexample, for equal moles of primer (10) and doubly labeled internalstandard, the TAMRA fluorescence from the internal standard is typically1.6× to 1.7× higher than the TAMRA fluorescence from the primer (10),suggesting that the FAM moiety transfers energy to the TAMRA dye in theinternal standard, resulting in artificially high TAMRA fluorescence.This measurement was used to correct the TAMRA fluorescence from theproduct (45) in each of the test sample lanes.

[0108] C. Reaction Conditions

[0109] The reaction conditions used to measure the bias that a mutantform of Taq DNA polymerase (“AmpliTaq FS”) shows for ddCTP overFAM-ddCTP are provided in the table below. (The numbers in parenthesisnext to certain components refer to elements in FIG. 3.) Component FinalConcentrations TRIS.Cl, pH 9.0 at 20° C. 80 mM 5′ TAMRA-Labeled Primer(10) 1000 nM Template (5) 1000 nM MgCl₂ 2.4 mM ddGTP (20) 200 μMFAM-ddCTP (25) 200 μM AmpliTaq FS Polymerase 8 nM Reaction Temperature60° C.

[0110] The assay reactions were prepared as follows. Two 2×-concentrated“Half Reaction Mixtures” were prepared and held on ice. A firstsolution, the “2× Enz-DNA” mixture, comprised 2000 nM template/primerDNA and 16 nM AmpliTaq FS in 80 mM TRIS buffer. A second solution, the“2× Mg·Nuc” mixture, comprised 80 mM TRIS buffer plus 4.8 mM MgCl₂ andeach of the nucleotides at 400 μM. A “Zero Time Control” sample wasprepared by adding 1 μl of the 2× Enz·DNA mixture to 25 μl of “STOPSolution” (0.5 M EDTA, ° C.), and, after mixing, adding 1 μl of the 2×Mg·Nuc mixture. The Zero Time Control sample was held on ice until theremainder of the timed samples were also collected for furtherprocessing.

[0111] The remainder of each Half Reaction Mixture was pre-incubated for5 minutes at 60° C., and the assay reaction was started by adding anequal volume of the 2× Mg·Nuc mixture to the 2× Enz·DNA mixture. Atappropriate time points (in this example, at 20 second intervals),samples were removed (2 μl each) and rapidly quenched in 25 μl of theice cold STOP Solution. A total of 10 samples were collected rangingover an elapsed time of about 200 s.

[0112] To prevent overloading of the detector in the Model 373 DNASequencer, samples were further processed to remove excessunincorporated FAM-ddCTP. This was accomplished by lithiumchloride-ethanol precipitation using tRNA as a carrier. 5 μl of aquenched sample was added to 250 μl of “PPT Solution” (consisting of 0.8M LiCl plus 0.2 μg/ml E. Coli tRNA). After mixing, 750 μl of 95% ethanolwas added. Each sample was mixed and held on ice for 30 to 60 minutes toprecipitate the primer/template DNA.

[0113] To prepare samples for loading onto the 373, the LiCI/Ethanolprecipitate was pelleted at 10,000×g in a microcentrifuge for 5 minutesand the supernatant fluid was removed by vacuum aspiration. After 5minutes air drying, 50 μl of “Gel Sample Solution” (50% formamide plus3% dextran blue) was added. Pellets were dissolved by vigorous mixingand heating at 95° C. for 3 minutes, after which 3 μl of each sample wasloaded into separate lanes of a 16% denaturing, polyacrylamidesequencing gel (ABI PRISM™ 373 DNA Sequencing System User's Manual).Electrophoresis running conditions were 2600 V, 50 mA, 100 mW. Detectionof the fluorescent signal in each of the bands was accomplished usingGeneScan™ software (PE Applied Biosystems, p/n 672-30).

[0114] D. Quantitation of Data

[0115] A “Control Lane” was loaded with 3 μl of Gel Sample Solutioncontaining 10 fmol of TAA-labeled primer and 10 fmol of doubledye-labeled internal standard. The amount of TAMRA fluorescence wasdetermined for the 25-mer band and compared to the TAMRA signal in theinternal standard band. In this case, as mentioned above, the TAMRAsignal from the internal standard was 1.6× higher than the TAMRA signalfrom the primer band. Therefore, the TAMRA signal in each of the assayproduct (45) bands was multiplied by a 1/1.6 correction factor.

[0116] The relative amounts of DNA in each of the bands for a given lanewas calculated by dividing the fluorescence units in each of the bandsby the total number of units for that lane and multiplying that figureby the concentration of the DNA primer/template in the reaction. Thisnormalized the signal in each of the lanes to the total DNAconcentration in the reaction and corrected for lane-to-lane variationdue to gel-loading artifacts.

[0117] To determine the rate of incorporation of each of thenucleotides, the amounts of DNA in each of the bands was plotted versustime and the linear portions of each curve were fitted using a linearleast squares fitting program. The rate of incorporation was calculatedfor the unlabeled ddC as the rate of appearance of the 26-mer assayproduct (40), while the rate of incorporation of FAM-ddC was calculatedas the rate of appearance of the apparent 27-mer assay product (45). Theratio of these rates represented the preference that the DNA polymeraseshowed for ddCTP over FAM-ddCTP.

[0118] E. Results

[0119]FIG. 4 shows representative data comparing the incorporation ratesof unlabeled and dye-labeled terminators. The data in the figure werecorrected for energy transfer effects as discussed above. The linkerused was the propargylamido linker.

[0120] The data in the table below indicate that there is a preferencefor unlabeled ddCTP over any of the dye-labeled derivatives testedirrespective of the particular dye or linker used. However, themagnitude of this preference is strongly dependent upon the type oflinker-arm used to attach the dye to the base. In the case of thetraditional propargylamido linker, ddCTP is preferred 65× more thanFAM-ddCIT and over 800× more than HEX-ddCT. When the linker arm isextended by only 3 atoms (an ether oxygen and two methylene carbons,i.e., the propargyl-1-ethoxyamido linker), the bias against FAM-ddCTP isreduced from 65× to 19× and for HEX-ddCTP from over 800× to only about4×, a decrease by a factor of over 200. Inserting two ether units (i.e.,the propargyl-2-ethoxyamido linker) between the propargyl linker and thedye, however, has a deleterious effect, increasing the bias againstFAM-ddCTP about 2-fold (from 65× to 110×). (Dye)-linkerddCTP/(Dye)-ddCTP (6-FAM)-propargylamido-C 65×(6-FAM)-propargyl-1-ethoxyamido-C 19× (6-FAM)-propargyl-2-ethoxyamido-C110×  (HEX)-propargylamido-C >800×    (HEX)-propargyl-1-ethoxyamido-C 4×

Example 5 Synthesis of5-{3-(2-Aminoethoxy)propyn-1-yl}-2′,3′-dideoxycytidine triphosphate (13)

[0121] A Materials and Methods

[0122] Thin layer chromatography (TLC) was conducted on glass platesprecoated with 250 μm layers of silica gel 60-F₂₅₄. Compounds werelocated on the TLC plate after developing by quenching of fluorescenceand/or by charring with 5% sulfuric acid. Flash column chromatographywas performed on SIP brand silica gel 60 Å, 230-400 Mesh ASTM (BaxterScientific p/n C4582-87). NMR spectra were obtained as follows: ¹H NMRspectra were recorded at 300 MMz on solutions in CDCl₃ (internal Me₄Si,δ0) or D₂O (external Me₄Si, δ0) at ambient temperature; ¹³C NMR spectrawere recorded at 75.5 MHz on solutions in CDCl₃ (internal Me₄Si, δ0);19F NMR spectra were recorded at 282.23 MHz on solutions in CDCl₃ or D₂O(external CFCl₃δ0); and ³¹P NMR spectra were recorded at 121.44 MHz onsolutions in D₂O. In all cases, NMR data were in accord with theproposed structures. Unless otherwise indicated, all reactions werecarried out at ambient temperature, and in the work-up, solutions inorganic solvents were washed with equal volumes of aqueous solutions.Organic solutions were generally dried over anhydrous Na₂SO₄ prior toconcentration on a rotary evaporator under vacuum with a bathtemperature of 40-50 ° C. The HPLC systems used for analytical andpreparative purposes were as follows:

[0123] Analytical reverse-phase HPLC: column: Spheri-5 RP-C18, 5 μmparticle size, 220×4.6 mm (PE Applied Biosystems p/n 0711-0017);gradient: 0 to 50% acetonitrile at 1.5.ml/min over 20 min, followed by50% acetonitrile to 100% acetonitrile at 1.5 ml/min over 10 min.

[0124] Analytical ion pair HPLC: column: Aquapore™ OD-300, 7 μm particlesize, 220×4.6 mm (PE Applied Biosystems p/n 0711-0331); gradient: 0 to40% acetonitrile at 1.5 ml/min over 30 min, followed by 40% acetonitrileto 60% acetonitrile at 1.5 ml/min over 5 min.

[0125] Preparative anion exchange HPLC: column: Aquapore™ Anion, 20 μmparticle size, 250×10 mm (PE Applied Biosystems p/n 0711-0172);gradient: 40% acetonitrile:60% 100 mM TEAB, pH 7.0 to 40%acetonitrile:60% 1.5 mM TEAB pH 8 at 4.5 ml/min over 20 min, followed byisocratic elution.

[0126] Preparative reverse phase HPLCC: column: Prep Nova Pak HR-C18, 6μm particle size, 60 Å pore size, 300×40 mm (Waters Division of theMillipore Corporation p/n WAT037704); gradient (for mono andtriphosphates): 100% 100 mM TEAB pH 7 to 20% acetonitrile: 80% 100 mMTEAB pH 7 at 50 ml/min over 30 min, followed by 20% acetonitrile: 80%100 mM TEAB pH 7 to 50% acetonitrile:50% 100 mM TEAB pH 7 over 10 min;gradient (for dye-labeled triphosphates): 100% 100 mM TEAB pH 7 to 10%100 mM TEAB pH 7: 90% acetonitrile.

[0127] B. Synthesis of 2-Phthalimidoethanol (3)

[0128] Potassium phthalimide 2 (2.7 g, 14.6 mmol) was added to asolution of bromoethanol 1 in N,N-dimethylformamide (12 mL, 14.1 mmol).After stirring for 12 h at 70° C., the mixture was concentrated and thendiluted with dichloromethane (100 mL). After removal of solids byfiltration, the organic layer was washed with water, dried, andconcentrated. The concentrate was purified by flash columnchromatography (3:2 to 2:3 hexane-ethyl acetate) to give compound 3 as awhite solid (1.19 g, 44.12%) having an R_(F) of 0.22 (3:2 hexane-ethylacetate). See FIG. 5.

[0129] C. Synthesis of 3-(2-Phthalimidoethoxy)propyne (5)

[0130] To a stirred solution of compound 3 (1.14 g, 5.96 mmol) inN,N-dimethylformamide (20 mL) was added NaH (0.36 g, 80%) dropwise.After complete NaH addition, stirring was continued for 0.5 h at roomtemperature, and then the reaction was cooled to 0° C. Propargyl bromide4 (1.5 mL, 13.47 mmol) was added, and the stirring was continued for anadditional 0.5 h at 0° C., then, at room temperature for 2 h. Aftercareful addition of methanol to decompose excess NaH, the solvent wasevaporated and the crude product was purified by flash columnchromatography (3:2 to 1:1 to 2:3 hexane-ethyl acetate) to give compound5 as a solid (495 mg, 36.2%) having an R_(F) of 0.22 (3:2 hexane-ethylacetate). See FIG. 5.

[0131] D. Synthesis of5-{3-(2-Phthalamidoethoxy)-propyn-1-yl}-2′,3′-dideoxycytidine (7)

[0132] 5-Iodo-2′,3′-dideoxycytidine 6 (100 ma, 0.3 mmol) was reactedwith compound 5 (158 mg, 0.69 mmol) in the presence of cuprous iodide(11.4 mg, 0.06 mmol), tetrakis(triphenylphosphine)palladium (69 mg, 0.06mmol), and triethylamine (84 μL, 0.6 mmol) in N,N-dimethylformamide (1mL) for 12 h at room temperature under Argon atmosphere. The reactionwas then diluted with 2 g bicarbonate form of Dowex-1 anion exchangeresin in methanol. After stirring for 1 h at room temperature thereaction mixture was filtered and concentrated. The product was purifiedby flash column chromatography (13:1 dichloromethane-methanol) to givecompound 7 (75 mg, 57.66%) having an R_(F) of 0.23 (solvent 9:1dichloromethane-methanol). See FIG. 6.

[0133] E. Synthesis of5-{3-(2-Trifluoroacetamidoethoxy)propyn-1-yl}-2′,3′-dideoxycytidine (8)

[0134] A mixture of compound 7 (73 mg, 0.17 mmol) and ethylenediamine(400 μL) was heated at 80° C. in ethanol (4 mL) for 1 h. The reactionwas then evaporated to dryness, the residue was dissolved inN,N-dimethylformamide (2 mL), and methyl trifluoroacetate (6.5 mL) wasadded. After stirring for 1 h at 80° C., the solvent was evaporated andthe residue was purified by flash column chromatography (9:1dichloromethane-methanol) to give compound 8 (36 mg, 50.7%) having anR_(F) of.0.24 (solvent 9:1 dichloromethane-methanol). See FIG. 6.

[0135] F. Synthesis of5-{3-(2′-Trifluoroacetamidoethoxy)propyn-1-yl}-2′,3′-dideoxycytidinemonophosphate (10)

[0136] Freshly distilled phosphorous oxychloride (16.2 μl, 0.17 mmol)was added to nucleoside 8 (18.8 mg, 0.046 mmol) in trimethylphosphate(150 μL) at −30° C. to form the corresponding dichloromonophosphate 9.The reaction mixture was allowed to warm to −5° C. over a period of 80minutes and stirring was continued for an additional 1 h at roomtemperature. The reaction was quenched with 2 M TEAB buffer pH 8.0, andpurified by preparative reverse phase HPLC as described above. Fractionscorresponding to product were concentrated to give monophosphate 10(12.3 mg, 54.56%). See FIG. 7.

[0137] G. Synthesis of5-{3-(2-Trifluoroacetamidoethoxy)propyn-1-yl}-2′,3′-dideoxycytidinetriphosphate (12)

[0138] The monophosphate 10 (7.4 mg, 15.3 mmol) dissolved inN,N-dimethylformamide (200 μL) was stirred with carbonyldiimidazole(CDI) (4.2 mg, 25.9 mmol) for 1 h at room temperature. Excess CDI wasquenched by the addition of dry methanol (40 μL). The activatedmonophosphate 11 was stirred with a solution of tributylammoniumpyrophosphate in N,N-dimethylformamide (160 μL) containingn-tributylamine (16 μL) for 24 h at room temperature. The reaction wasquenched with 2 M TEAB pH 8.0 and purified by preparative reverse phaseEPLC as described above. The fractions corresponding to product wereconcentrated to give triphosphate 12. See FIG. 8.

[0139] H. Synthesis of5-{3-(2-Aminoethoxy)propyn-1-yl}-2′,3′-dideoxycytidine triphosphate (13)

[0140] The purified protected triphosphate 12 was taken up inconcentrated aqueous NH₄H (4 mL) and stirred for 2.5 h at roomtemperature. The reaction mixture was concentrated to give compound 13which was formulated with 0.1 M TEAB pH 7.0 to a concentration of 2.6mM. The concentration and purity of the formulated bulk were confirmedby UV/Vis spectroscopy and analytical ion pair HPLC as described above,respectively. See FIG. 8.

Example 6 Synthesis of5-[3-{2-(2-Aminoethoxy)ethoxy}propyn-1-yl]-2′,3′-dideoxycytidinetriphosphate (23)

[0141] A. Materials and Methods

[0142] The materials and methods were essentially the same as describedabove with respect to Example 5.

[0143] B. Synthesis of 2-(2-Phthalimidoethoxy)ethanol (15)

[0144] To a solution of 2-(2-chloroethoxy)ethanol 14 (5 mL, 47.4 mmol)in N,N-dimethylformamide (35 mL) was added potassium phthalimide 2 (8.8g, 47.51 mmol), and the reaction mixture was stirred for 20 h at 70° C.The mixture was concentrated and then diluted with dichloromethane (300mL), and the organic layer was washed with water, dried, andconcentrated. The residue was purified by flash column chromatography(3:2 to 2:3 hexane-ethyl acetate) to give compound 15 as a white solid(7.15 g, 64.17%) having an R_(F) of 0.12 (solvent 3:2 hexane-ethylacetate). See FIG. 9.

[0145] C. Synthesis of 3-[2-(2-Phthalimidoethoxy)]ethoxypropyne (16)

[0146] To a stirred solution of compound 15 (1.39 g, 5.91 mmol) inN,N-dimethylformamide (25 mL) was added NaH (0.33 g, 80%) dropwise.After complete NaH addition, stirring was continued for 0.5 h at roomtemperature and then cooled to 0 ° C. Propargyl bromide 4 (1.24 mL,11.13 mmol) was added, and stirring was continued for 0.5 h at 0° C.,then for 2 h at room temperature. After careful addition of methanol todecompose excess NaH, the solvent was evaporated and crude product waspurified by flash column chromatography (3:2 to 1:1 to 2:3 hexane-ethylacetate) to give compound 16 as a solid (591 mg, 36.6%) having an R_(F)of 0.41 (solvent 3:2 hexane-ethyl acetate). See FIG. 9.

[0147] D. Synthesis of5-[3-{2-(2-Phthalimidoethoxy)ethoxy}propyn-1-yl]-2′,3′-dideoxycytidine(17)

[0148] 5-Iodo-2′,3′-dideoxycytidine 6 (240 mg, 0.71 mmol)) was reactedwith compound 16 (810 mg., 2.96 mmol) in the presence of cuprous iodide(33 mg, 0.173 mmol), tetrakis(triphenylphosphine)palladium (164 mg,0.142 mmol), and triethylamine (198 μL, 1.42 mmol) inN,N-dimethylformamide (4 mL) for 12 h at room temperature under Argonatmosphere. The reaction was then diluted with 4 g bicarbonate form ofDowex 1 anion exchange resin in methanol. After stirring for 1 h at roomtemperature the reaction mixture was filtered and concentrated. Theproduct was purified by flash column chromatography (13:1dichloromethane-methanol) to give compound 17 (245 mg, 71.3%) having anR_(F) of 0.35 (solvent 9:1 dichloromethane-methanol). See FIG. 10.

[0149] E. Synthesis of5-[3-{2-(2-Trifluoroacetamidoethoxy)ethoxy}-propyn-1-yl]2′,3′-dideoxycytidine(18)

[0150] A mixture of compound 17 (230 mg, 0.48 mmol) and ethylenediamine(1 mL) was heated at 80° C. in ethanol (10 mL) for 1 h. The reactionmixture was then evaporated to dryness, the residue was dissolved inN,N-dimethylformamide (5 mL), and methyl trifluoroacetate (15 mL) wasadded. After stirring for 1 h at 80° C., solvent was evaporated andresidue was purified by flash column chromatography (13:1dichloromethane-methanol) to give compound 18 (72 mg, 33.7%) having anR_(F) of 0.37 (solvent 9:1 dichloromethane-methanol). See FIG. 10.

[0151] F. Synthesis of5-[3-{2-(2-Trifluoroacetamidoethoxy)ethoxycypropy}-1-yl]-2′,3′-dideoxycytidinemonophosphate (20)

[0152] Freshly distilled phosphorous oxychloride (34.8 μl, 369 μmol) wasadded to nucleoside 18 (41.4 mg, 92 μmol) in trimethylphosphate (350 μL)at −30° C. to form the corresponding dichloromonophosphate 19. Thereaction mixture was allowed to warm to −5° C. over a period of 80minutes and stirring was continued for 1 h at room temperature. Thereaction was quenched with 2 M TEAB pH 8.0 buffer and purified bypreparative reverse phase HPLC as described above. The fractionscorresponding to product were concentrated to give monophosphate 20(14.8 mg, 27.6%). See FIG. 11.

[0153] G. Synthesis of5-[3-{2-(2-Trifluoroacetamidoethoxy)ethoxy}-propyn-1-yl]-2′,3′-dideoxycytidinetriphosphate (22)

[0154] The monophosphate 20 (14.8 mg, 28 μmol) dissolved inN,N-dimethylformamide (300 μL) was stirred with carbonyldiimidazole(CDI) (13.5 mg, 83.26 μmol) for 1 h at room temperature. Excess CDI wasquenched by the addition of dry methanol (80 μL). The activatedmonophosphate 21 was stirred with a solution of tributylammoniumpyrophosphate (160 mg) in N,N-dimethylformamide (300 μL) containingn-tributylamine (32 μL) for 24 h at room temperature. The reaction wasquenched with 2 M TEAB pH 8.0 and purified by preparative reverse phaseHPLC as described above. The fractions corresponding to product wereconcentrated to give triphosphate 22 (0.9 mg, 4.7%). See FIG. 12.

[0155] H. Synthesis of5-[3-{2-(2-Aminoethoxy)ethoxy}propyn-1-yl]-2′,3′-dideoxycytidinetriphosphate (23)

[0156] The purified protected triphosphate 22 was taken up inconcentrated aqueous NH₄₀H (2 mL) and stirred for 3 h at approximately48° C. The reaction mixture was concentrated to give compound 23 whichwas formulated with 0.1 M TEAB pH 7.0 to a concentration of 3.5 mM. Theconcentration and purity of the formulated bulk were confirmed by UV/Visspectroscopy and analytical ion pair HPLC as described above,respectively. See FIG. 12.

Example 7 Attachment of Dye to5-{3-(2-aminoethoxy)propyn-1-yl}-2′,3′-nucleotide

[0157] The nucleoside aminotriphosphate in 100 mM TEA-bicarbonate (pH7.0) was evaporated to dryness. It was then resuspended in 250 mMbicarbonate buffer (pH 9.0). A solution of Dye-NHS (in DMSO) was addedand stirred in the dark overnight at room temperature. The reactionmixture was purified by preparative anion exchange HPLC as describedabove. The fractions corresponding to product were concentrated andrepurified by preparative reverse phase HPLC as described above. Finalproduct was dried in vacuo and diluted with 50 mM CAPSO, pH 9.6, to aconcentration of 1 mM. The concentration and purity of the formulatedbulk is confirmed by TV/VIS spectroscopy and analytical ion-pairing HPLCas described above, respectively.

Example 8 Improved Peak Height Evenness Using the PropargylethoxyaminoDideoxynucleotides of the Invention

[0158]FIG. 13 shows single color sequencing reactions using dye-labeledddCTP as the terminator. The top panel shows results using aDTAMRA-1-labeled terminator using a propargylarido linker, while thebottom pannel shows results using a DTAMRA-2-labeled terminator using anpropargyl-1-ethoxyamido linker of the present invention. Different dyeisomers were used to produce optimum results—the 1 isomer being thepreferred compound for use with the propargylamido linker and the 2isomer being the preferred compound for use with thepropargyl-1-thoxyamido linker. The portion of the sequencing laddershown in FIG. 13 starts with a pair of C's at bases 495 and 496 and endswith single Cs at bases 553, 557 and 560 in the sequence of pGEM-3ZI(+)using the -21 M13 Primer (forward). In the top panel, in the group of 6C's, the first C is present only as a leading shoulder rather than adistinct peak, while in the bottom panel, all 6 Cs are clearly resolved.The resolution of the 6 Cs in the bottom panel is made possible by themore even peak heights which are possible using thepropargyl-1-ethoxyamido linker of the present invention in combinationwith rhodamine-type dyes. This enhanced resolution of neighboring peaksfacilitates automated base-calling routines used in automated DNAsequencing systems.

[0159] All publications and patent applications are herein incorporatedby reference to the same extent as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference.

[0160] Although only a few embodiments have been described in detailabove, those having ordinary skill in the chemical arts will clearlyunderstand that many modifications are possible in the preferredembodiment without departing from the teachings thereof All suchmodifications are intended to be encompassed within the followingclaims.

We claim:
 1. A nucleoside compound having the structure:

wherein: R₁ and R₂ taken separately are selected from the groupconsisting of —H lower alkyl protecting group, and label; B is a7-deazapurine, purine, or pyrimidine nucleoside base; wherein when B ispurine or 7deazapurine, the sugar moiety is attached at the N⁹-positionof the purine or deazapurine, and when B is pyrimidine, the sugar moietyis attached at the N¹-position of the pyrimidine; and wherein when B isa purine, the adjacent triple-bonded carbon is attached to the8-position of the purine, when B is 7-deazapurine, the adjacenttriple-bonded carbon is attached to the 7-position of the 7-deazapurine,and when B is pyrimidine, the adjacent triple-bonded carbon is attachedto the 5-position of the pyrimidine; W₁ is selected from the groupconsisting of —H and ——OH; W₂ is —OH or a moiety which renders thenucleoside incapable of forming a phosphodiester bond at the3′-position; and W₃ is selected from the group consisting of —PO₄,—P₂O₇, —P₃O₁₀, phosphate analog, and —OH.
 2. The nucleoside compound ofclaim 1 wherein one of R₁ and R₂ is label.
 3. The nucleoside compound ofclaim 2 wherein the label is a fluorescein-type dye.
 4. The nucleosidecompound of claim 2 wherein the label is a rhodamine-type dye.
 5. Thenucleoside compound of claim 1 wherein W₁ is —H and W₂ is —OH or amoiety, and W₃ is —P₃O₁₀.
 6. The nucleoside compound of claim 1 whereinW₁ is —H, W₂ is —OH or a moiety, and W₃ is —P₃O₁₀.
 7. The nucleosidecompound of claim 1 wherein W₂ is —OH or a moiety selected from thegroup consisting of —H, azido, amino, fluro, and methoxy.
 8. Thenucleoside of claim 1 wherein B is selected from the group consisting ofuracil, cytosine, 7-deazaadenine, and 7-deazaguanosine.
 9. A method forperforming a primer extension reaction comprising the steps of:providing a template nucleic acid; annealing an oligonucleotide primerto a portion of the template nucleic acid; and adding primer-extensionreagents to the primer-template hybrid for extending the primer, theprimer extension reagents including a nucleoside compound having thestructure:

wherein: R₁ and R₂ taken separately are selected from the groupconsisting of —H lower alkyl, protecting group, and label; B is a7-deazapurine, purine, or pyrimidine nucleoside base; wherein when B ispurine or 7-deazapurine, the sugar moiety is attached at the N⁹-positionof the purine or deazapurine, and when B is pyrimidine, the sugar moietyis attached at the N¹-position of the pyrimidine; and wherein if B is apurine, the adjacent triple-bonded carbon is attached to the 8-positionof the purine, if B is 7-deazapurine, the adjacent triple-bonded carbonis attached to the 7-position of the 7-deazapurine, and if B ispyrimidine, the adjacent triple-bonded carbon is attached to the5-position of the pyrimidine; W₁ is selected from the group consistingof —H and —OH; W₂ is H or a moiety which renders the nucleosideincapable of forming a phosphodiester bond at the 3′-position; and W₃ isselected from the group consisting of —PO₄, —P₂O₇, —P₃O₁₀, phosphateanalog, and —OH.
 10. The method of claim 9 wherein one of R, and R₂ islabel and the other is —H.